Vibrio cholerae is the causative agent of the diarrheal disease cholera, a debilitating disease affecting as many as ten million people annually. The disease is caused by cholera toxin, a protein whose expression is regulated by two membrane-localized transcription factors, ToxR and TcpP. This application investigates how ToxR and TcpP combine to activate the toxT promoter in V. cholerae, thus triggering virulence gene expression. In addition, we will investigate how ToxR activates a second promoter, ompLJ, in a TcpP- independent fashion. Expression of the porin, OmpU, leads to bile resistance in V. cholerae. We hypothesize that ToxR functions by different mechanisms at the ompU and toxT promoters. With ToxR directly activating ompU while serving to facilitate TcpP-mediated promoter activation at toxT. ' Finally, we will also develop a system for observing the the interaction of ToxR and TcpP in the membrane of living V. cholerae cells using FRET. This will allow us to observe a critic step in induction of virulence in \ real time as well as allow us to observe any changes in ToxR/TcpP interaction in response to a variety of environmental conditions. Specific Aims of this project are: I. Determine the mechanism of ompU and toxT activation by ToxR II. Determine the mechanisms of DNA-binding, ToxR interaction and RNA polymerase stimulation by TcpP and the role of each event in toxT activation III. Measure ToxR/TcpP protein-protein interactions in living V. cholerae cells after tcpP induction using FRET (fluorescence resonance energy transfer) [unreadable] [unreadable] [unreadable]